1. Field of the Invention
The invention pertains to extracts of Picralima nitida seeds, fruit rind and stem bark, and the use of these extracts in the treatment of malaria, leishmaniases and trypanosomosis.
The alkaloid extracts of the fruits of P. nitida exhibit activity against drug-resistant and drug-sensitive malarial strains of Plasmodium falciparum and these alkaloids show significant inhibitory activity against both clones of P. falciparum at IC.sub.50 values of 0.0.1-0.9 .mu.g/ml.
The invention also pertains to the use of methanol extracts from Picralima nitida for use in the treatment of leishmaniases.
2. Description of the Prior Art
Picralima nitida (Fam. Apocynaceae) is the source of the bitter tasting "Akuamma" seeds, employed extensively in west Africa as an ingredient in many folk remedies.sup.1,.sup.2. The aqueous extract of the bark and seeds are used for the treatment of malaria and pyrexia, and the powdered seeds have been dispensed as a cure for pnumonia and other infections.sup.3. FNT .sup.1 Irvine, F. R. (1961) Woody Plants of Ghana, Oxford University Press, London. pp.629-630. FNT .sup.2 Oliver-Bever, B. (1982) Medicinal plants in tropical west Africa 1. Plants acting on the cardiovascular system, J. Ethnopharmacol 5, 1-72. FNT .sup.3 Ainslie, J. R. (1937) List of Plants Used in Native Medicine in Nigeria, Imperial Forestry Institute, Oxford. p.71.
The plant Picralima nitida contains several indole and dihydroindole alkaloids, of which the major ones include akuammiline, akuammidine, akuammine, akuammigine, akuammicine, picraline and picraphylline.sup.4,.sup.5. The principal alkaloid found in the plant, akuammine, has been shown to be inactive against avian malaria and in clinical trials.sup.6. FNT .sup.4 Saxon, J. E. (1973) Alkaloids of Picralima and Alstonia species. In: R. H. F. Manske (ed.), The alkaloids - Chemistry and physiology. Academic Press, New York. p. 157-159. FNT .sup.5 Ansa-Asamoah, R., Kapadia, G. J., Lloyd, H. A. and Sokoloski, E. A. (1990) Picratidine, A new indole alkaloid from Picralima nitida seeds, J. Nat. Prod., 53(4), 975-977. FNT .sup.6 Paris, R. and Moyse, H. (1971) Precis de Matiere Medicale, vol.3, Masson, Paris, cited in Bep-Oliver, op cit.
Akuammine, however, is a strong sympathicomimetic and possesses local anesthetic action comparable to that of cocaine.sup.7. FNT .sup.7 Raymond-Hamet, R. (1951) Sur une drogue remarquable de l'Afrique tropicale, le Picralima nitida (stapf) Th. & R. Dur. Revue de Botanique. Appliquee 31, 465.
Another major Picralima alkaloid, akuammidine has been shown to possess a strong local anaesthetic action and was found to be three times as active as cocaine hydrochloride.sup.8. The compound also has sympatholytic and a mild, but persistent, hypotensive effect. Extracts of the plant have been shown to posses significant analgesic activity in the rat pedal model.sup.9. The hot water decoction of the stem bark has been shown to possess significant in vivo activity against Trypanosoma brucei in rats, and the activity was found comparable to the effect of 8 mg kg.sup.-1 of dimenazene aceturate (Wosu and Ibe, 1989). A CNS active indole alkaloid, pericine, has been detected by opiate receptor binding studies from the cell suspension culture of P. nitida.sup.10. Although seeds and stem bark of P. nitida are employed as aqueous ethanol (palm wine) decoctions in the treatment of severe cases of malaria in Nigeria, Ghana and many parts of Africa, there is presently no scientific investigation to support the use of the herb as a malaria remedy. FNT .sup.8 Raymond-Hamet, R. (1944) Picralima nitida. These Doc. Pharm., Paris. FNT .sup.9 Ezekwesili, J. (1983) Pharmacological activity of Picralima nitida, M. Pharm Dissertation, University of Nigeria, Nsukka. FNT .sup.10 Arens, H., Borbe, H. O. Ulbrich, B. and Stockigt, J. (1982), Detection of pericine, a new CNS-active indole alkaloid, Planta Med. 46(4). pp. 210-214.
Infections due to protozoa of the genus Leishmania are a major world-wide health problem, with high endemicity in developing countries, however, the global prevalence of leishmaniases in man is about 12 million cases, with an estimated incidence of 2-3 million cases per annum.sup.11. The pathological effects of the disease are complex and manifests in various forms ranging from self-healing cutaneous lesions; recurrent leishmaniasis recidivans; disfiguring mucocutaneous and diffuse cutaneous diseases; to fatal systemic infection, visceral leishmaniases or kala azar. In the later form, the reticuloendoethelial system is infected with the resultant toll on the spleen, liver, bone marrow, lymph glands, and, often, some degree of intestinal tract dysfunction. Approximately 350 million people within 80 countries are threatened by the disease worldwide. FNT .sup.11 World Report on Tropical Diseases. WHO Features 139 (March, 1990): 1-12.
Unfortunately, clinical drug intervention is presently limited to the use of pentavalent antimonials (SbV), sodium stilbogluconate and N-methylglucamine antimonate, and, secondarily, amphotericin or pentamidine.sup.12,.sup.13. These antileishmanials require parenteral administration with clinical supervision or hospitalization during treatment because of the severity of possible toxic side-effects that include cardiac and/or renal failure.sup.14. FNT .sup.12 Bryceson, A. (1987) Therapy in man. Peters, W., Killick-Kendrick, R., eds., The Leishmaniases in Biology and Medicine, Vol. 2, New York: Academic Press, pp. 847-907. FNT .sup.13 Croft, S. L. (1988) Recent developments in the chemotherapy of leishmaniasis. Trends Pharmacol Sci 9:376-381. FNT .sup.14 Bryceson, A. (1987) Therapy in man. Peters, W., Killick-Kendrick, R., eds, The Leishmaniases in Biology and Medicine, Vol. 1, New York: Academic Press, pp. 847-907.
Treatment with the aforementioned agents is not consistently effective particularly for the most virulent leishmanial disease forms.sup.15,.sup.16,.sup.17,.sup.18. The World Health Organization has reported large scale resistance of kala azar to SbV, which are the preferred chemotherapy for treatment of most forms of leishmanial disease (TDR News, Dec., 1990). In some endemic regions, it has been observed that prolonged medication (22 months or more) with SbV is required to effect a clinical cure.sup.19. Long term SbV therapy, however, is not usually advocated due to the mentioned cardiac and renal toxicity of SbV. FNT .sup.15 Jha, T. K. (1983) Evaluation of diamidine compound (pentamidine isethionate) in the treatment of resistant cases of kala azar occurring in North Bihar, India. Trans Roy Soc Trop Med Hyg 77:167-170. FNT .sup.16 Rocha, R. A., Sampaio, R. N., Guerra, M., Magalhaes, A., Cuba, C. C., Barreto, A. C., Marsden, P. D. (1980) Apparent Glucantime failure in five patients with mucocutaneous leishmaniasis. J Trop Med Hyg 83:131-139. FNT .sup.17 Mebrahtu, Y. B., Lawyer, P., Githure, J., Were, J. B., Muigai, R., Hendricks, L., Leeuwenburg, J., Koech, D., Roberts, C. (1989) Visceral leishmaniasis unresponsive to pentostam caused by Leishmania tropica in Kenya. Am J Trop Med Hyg 41:289-294. FNT .sup.18 Anonymous (1990) Antimonials: large-scale failure in leishmaniasis "alarming". Trop Dis Rsch News (World Health Organization Special Program for Research and Training in Tropical Diseases) 34:1&7. FNT .sup.19 Bryceson, A. (1987) Therapy in man. Peters, W., Killick-Kendrick, R., eds, The Leishmaniases in Biology and Medicine, Vol. 2, New York: Academic Press, pp. 847-907.
There is, therefore, a need for the development of more effective, less toxic and orally active antileishmanial agents. However, development of a new drug for the treatment of leishmaniasis has been impeded by the lack of a simple, rapid and universally applicable (to the various Leishmania species/strains infecting humans) drug evaluation system.sup.20,.sup.21. The lack of progress in the development of new antileishmanial agents is evident by the fact that all the clinically useful drugs were developed between 1947 and 1959.sup.22. Current methods for screening potential antileishmanial agents generally utilize intracellular amastigotes (the mammalian intracellular form) since promastigotes (monoflagellate forms found within the insect vector and culture in vitro) are reported "insensitive" within in vitro assays to SbV compounds used for human leishmaniases.sup.23. Since there is no system yet available for culturing amastigotes extracellularly except re-isolation from infected tissues and macrophage cultures, their mass culture is rather limited.sup.24,.sup.25, thereby making them unsuitable for primary screening of potential antileishmanial agents. FNT .sup.20 Croft, S. L. (1986) In vitro screens in the experimental chemotherapy of leishmaniasis and trypanosomiasis. Parasit Today 2:64-69. FNT .sup.21 Neal, R. A. (1987) Experimental chemotherapy. Peters, W., Killick-Kendrick, R., eds, The Leishmaniases in Biology and Medicine, Vol 2, New York: Academic Press, pp. 793-845. FNT .sup.22 Jackson, J. E., Tally, J. D., Tang, D. B. (1989) An in vitro micromethod for drug sensitivity testing of Leishmania. Am J Trop Med Hyg 41:318-330. FNT .sup.23 Ibid. FNT .sup.24 Ibid. FNT .sup.25 Croft, S. L. (1986) In vitro screens in the experimental chemotherapy of leishmaniasis and trypanosomiasis. Parasit Today 2:64-69.
An in vitro radiorespirometric microtest using promastigotes has been developed which relies on drug inhibition of parasite production of .sup.14 CO.sub.2 03 +3* U .sup.W Etery of .sup.14 C-substrates by promastigotes to detect drug-mediated parasite damage at low drug concentration within a short time.sup.26,.sup.27. The test is quantitative, rapid, consistent, and conducted in a serum-free chemically defined medium in which prior adaptation is not necessary to cultivate the so-called "difficult to grow" species. The method has been shown to correlate to patients response to SbV therapy.sup.28. FNT .sup.26 Jackson, J. E., Tally, J. D., Tang, D. B. (1989) An in vitro micromethod for drug sensitivity testing of Leishmania. Am J Trop Med Hyg 41:318-330. FNT .sup.27 Jackson, J. E., Tally, J. D., Ellis, W. Y., Membrahtu, Y. B., Lawyer, P. G., Were, J. B., Reed, S. G., Panisko, D. M., Limmer, B. L. (1990) Quantitative in vitro drug potency and drug susceptibility evaluation of leishmania spp. from patients unresponsive to pentavelent antimony therapy. Am J Trop Med Hyg 43:464-480. FNT .sup.28 Ibid.
Visceral leishmaniasis is endemic to the central Nigerian highlands, and zoonotic cutaneous leishmaniasis, prevalent in the northern half of this country. Therefore, because of limited supply, expense and toxicity of commercial antileishmanials, traditional herbal therapy is frequently utilized in many leishmanial endemic regions of Nigeria.